We have demonstrated the RNA interference-based interruption of cellular controllers to increase recombinant protein yield in Drosophila Schneider 2 (S2) cell culture. Double-stranded RNA (dsRNA) was enzymatically synthesized in vitro and transfected into stable cell lines expressing green fluorescent protein (GFP) under an inducible promoter. Components of cell cycling (CycE and ago) were silenced with dsRNA homologous to a 700-nucleotide section of their respective mRNA transcripts. Silencing ago and CycE resulted in increases in product yield of up to 1.8-fold and 4-fold, respectively, relative to a control transfected with nuclease-free water. It is surprising to note that nearly complete silencing of CycE resulted in no significant change in GFP fluorescence after 24 h, and a decrease in fluorescence after 72 h. By partially silencing CycE, however, we were able to retain 80% of the cells in G1 (48-h sample) and increase GFP synthesis by fourfold. Implications for protein synthesis processing are discussed.
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Abu-Absi NR, Srienc F (2000) Cell cycle events and cell cycle dependent processes in eukaryotic cells. In: Spier RE (ed) Encyclopedia of cell technology. Wiley-Interscience, New York, pp 320–383
Boutros M, Kiger AA, Armknecht S, Kerr K, Hild M, Koch B, Haas SA, Consortium HF, Paro R, Perrimon N (2004) Genome-wide RNAi analysis of growth and viability in Drosophila cells. Science 303(5659):832–835
Brumby AM, Zraly CB, Horsfield JA, Secombe J, Saint R, Dingwall AK, Richardson H (2002) Drosophila Cyclin E interacts with components of the Brahma complex. EMBO J 21(13):3377–3389
Clemens JC, Worby CA, Simonson-Leff N, Muda M, Maehama T, Hemmings BA, Dixon JE (2000) Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc Natl Acad Sci USA 97(12):6499–6503
Fussenegger M, Bailey JE (1998) Molecular regulation of cell-cycle progression and apoptosis in mammalian cells: implications for biotechnology. Biotechnol Prog 14(6):807–833
Fussenegger M, Schlatter S, Datwyler D, Mazur X, Bailey JE (1998) Controlled proliferation by multigene metabolic engineering enhances the productivity of Chinese hamster ovary cells. Nat Biotechnol 16(5):468–472
Hacker DL, Bertschinger M, Baldi L, Wurm FM (2004) Reduction of adenovirus E1A mRNA by RNAi results in enhanced recombinant protein expression in transiently transfected HEK293 cells. Gene 341:227–234
Ibarra N, Watanabe S, Bi JX, Shuttleworth J, Al-Rubeai M (2003) Modulation of cell cycle for enhancement of antibody productivity in perfusion culture of NS0 cells. Biotechnol Prog 19(1):224–228
Jia J, Zhang W, Wang B, Trinko R, Jiang J (2003) The Drosophila Ste20 family kinase dMST functions as a tumor suppressor by restricting cell proliferation and promoting apoptosis. Genes Dev 17(20):2514–2519
Jones L, Richardson H, Saint R (2000) Tissue-specific regulation of Cyclin E transcription during Drosophila melanogaster embryogenesis. Development 127(21):4619–4630
Kramer SF, Bentley WE (2003) RNA interference as a metabolic engineering tool: potential for in vivo control of protein expression in an insect larval model. Metab Eng 5(3):183–190
Lee LA, Orr-Weaver TL (2003) Regulation of cell cycles in Drosophila development: intrinsic and extrinsic cues. Annu Rev Genet 37:545–578
Maciorowski Z, Delic J, Padoy E, Klijanienko J, Dubray B, Cosset JM, Dumont J, Magdelenat H, Vielh P (1998) Comparative analysis of apoptosis measured by Hoechst and flow cytometry in non-Hodgkin’s lymphomas. Cytometry 32(1):44–50
March JC, Bentley WE (2004) Insulin stimulates double-stranded RNA uptake in Drosophila S2 cells. Biotechniques 37(6):890–898
Mazur X, Fussenegger M, Renner WA, Bailey JE (1998) Higher productivity of growth-arrested Chinese hamster ovary cells expressing the Cyclin-dependent kinase inhibitor p27. Biotechnol Prog 14(5):705–713
Mazur X, Eppenberger HM, Bailey JE, Fussenegger M (1999) A novel autoregulated proliferation—controlled production process using recombinant CHO cells. Biotechnol Bioeng 65(2):144–150
Moberg KH, Bell DW, Wahrer DC, Haber DA, Hariharan IK (2001) Archipelago regulates Cyclin E levels in Drosophila and is mutated in human cancer cell lines. Nature 413(6853):311–316
Qu ZL, MacLellan WR, Weiss JN (2003a) Dynamics of the cell cycle: checkpoints, sizers, and timers. Biophys J 85(6):3600–3611
Qu ZL, Weiss JN, MacLellan WR (2003b) Regulation of the mammalian cell cycle: a model of the G(1)-to-S transition. Am J Physiol Cell Physiol 284(2):C349–C364
Somasundaram K, Zhang HB, Zeng YX, Houvras Y, Peng Y, Zhang HX, Wu GS, Licht JD, Weber BL, ElDeiry WS (1997) Arrest of the cell cycle by the tumour-suppressor BRCA1 requires the CDK-inhibitor p21(WAF1/CiP1). Nature 389(6647):187–190
Suzuki E, Ollis DF (1989) Cell-cycle model for antibody-production kinetics. Biotechnol Bioeng 34(11):1398–1402
Suzuki E, Ollis DF (1990) Enhanced antibody-production at slowed growth-rates—experimental demonstration and a simple structured model. Biotechnol Prog 6(3):231–236
Watanabe S, Shuttleworth J, Al-Rubeai M (2002) Regulation of cell cycle and productivity in NS0 cells by the over-expression of p21(CIP1). Biotechnol Bioeng 77(1):1–7
The authors express gratitude to Dr. David Mosser at the University of Maryland for help with flow cytometry. This work was funded by NIH grant 1R01GM70851-01.
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March, J.C., Bentley, W.E. RNAi-based tuning of cell cycling in Drosophila S2 cells—effects on recombinant protein yield. Appl Microbiol Biotechnol 73, 1128–1135 (2007). https://doi.org/10.1007/s00253-006-0560-x
- Cell culture
- Cyclin E
- Cell cycle